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Abstract

Background

Plant viral vectors offer high-yield expression of pharmaceutical and commercially
important proteins with a minimum of cost and preparation time. The use of Agrobacterium tumefaciens has been introduced to deliver the viral vector as a transgene to each plant cell
via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation,
a full length, systemically moving virus is no longer necessary for excellent protein
yield, since the viral transgene is transcribed and replicates in every infiltrated
cell. Viral genes may therefore be deleted to decrease the potential for accidental
spread and persistence of the viral vector in the environment.

Results

In this study, both the coat protein (CP) and triple gene block (TGB) genetic segments
were eliminated from Foxtail mosaic virus to create the "FECT" vector series, comprising a deletion of 29% of the genome. This
viral vector is highly crippled and expresses little or no marker gene within the
inoculated leaf. However, when co-agroinoculated with a silencing suppressor (p19
or HcPro), FECT expressed GFP at 40% total soluble protein in the tobacco host, Nicotiana benthamiana. The modified FoMV vector retained the full-length replicase ORF, the TGB1 subgenomic
RNA leader sequence and either 0, 22 or 40 bases of TGB1 ORF (in vectors FECT0, FECT22
and FECT40, respectively). As well as N. benthamiana, infection of legumes was demonstrated. Despite many attempts, expression of GFP
via syringe agroinoculation of various grass species was very low, reflecting the
low Agrobacterium-mediated transformation rate of monocots.

Conclusions

The FECT/40 vector expresses foreign genes at a very high level, and yet has a greatly
reduced biohazard potential. It can form no virions and can effectively replicate
only in a plant with suppressed silencing.

Background

Plant expression systems have been developed as production platforms for many therapeutic
proteins over the past two decades. Although many foreign proteins have been expressed
in stably transgenic plants, plant viral vectors have emerged as the most efficient
approach to achieving high-level expression of recombinant proteins in plants [1,2]. These self-replicating vectors produce maximum levels of foreign gene expression
and require minimal set-up time. It is often possible to generate purified recombinant
protein within three weeks of receiving a gene sequence [3,4].

However, the potential widespread use of recombinant viruses raises concerns about
possible risks to the environment. Bio-safety issues must be considered to prevent
the spread of the genetically engineered virus from experimental plants to susceptible
wild plants [5-7]. Intact viral vectors have the potential to spread and infect non-target plants,
but replication-defective or movement-defective viruses avoid these problems. These
deleted viral vectors also address cross-contamination issues in the growth room and
greenhouse. In the field, it might be possible to achieve high expression in transgenic
plants carrying an inducible virus as a transgene [8,9]. In all of these cases, deleted virus vectors would be greatly preferred over full
virus vectors for reduced transmission and persistence.

An obvious disadvantage to the deleted virus approach is that the vector cannot spread
past the originally inoculated cells. However, this weakness can be successfully overcome
by the agroinoculation technique, which uses Agrobacterium tumefaciens to deliver the virus sequence, carried in a binary vector, to the genome of the vast
majority of plant cells in the infiltration zone of the leaf using whole, nonsterile
plants [10]. For small scale use, a syringe is used to infiltrate leaves with Agrobacterium, while for large scale applications, vacuum infiltration is used to inoculate an
entire greenhouse at once [10]. For both agroinoculation and transgenic use, systemic spread becomes an unnecessary
property. Agroinoculation involves the local transformation of the infiltrated leaf
with the viral cDNA as a part of the T-DNA of the Ti plasmid. A plant promoter (most
commonly CaMV 35S) placed upstream of the viral cDNA induces the transcription of
viral genome in the plant nucleus and viral RNA is transported to cytoplasm for viral
replication.

Over the past few years, several deleted viral vectors delivered by agroinoculation
have been created and some are used commercially. Tobacco mosaic virus (TMV) lacking the coat protein (CP) gene has been used to express a large number of
foreign proteins commercially [4,11,12]. Removal of the CP gene from TMV can lead to unexpectedly large increases in foreign
gene expression [13]. In the Potato virus X (PVX) replacement virus vector, both the triple gene block (TGB) and CP viral genes
were removed, leaving only the replicase gene and terminal untranslated regions, and
these deleted genes were replaced with GFP [14]. The expression level of GFP from this vector was 2.5-fold higher than that of full-length
PVX vector with the GFP encoding sequence between the triple gene block and the CP
genes. A defective RNA TMV vector has also been shown to express at high levels [15].

Agrobacterium infiltration-mediated transient expression can be greatly enhanced by suppression
of gene silencing. An RNA silencing suppressor, such as p19 [16] from tomato bushy stunt virus or HcPro [17] from potato virus Y, is co-inoculated in a separate strain of Agrobacterium along with the Agrobacterium carrying the viral cDNA. Using this approach, highly efficient production of GFP from
a TMV-based vector was achieved with up to a 100-fold increase of the overexpression
level [18]. As well, potexvirus expression was greatly increased with suppressor co-inoculation
[14].

The FECT vectors are derived from foxtail mosaic virus (FoMV) which is a member of
the genus Potexvirus. Potexvirus is a large group of flexuous and filamentous plant viruses with a single-stranded,
positive-sense genomic RNA which has a cap structure at the 5' terminus and a poly-(A)
tail at the 3' terminus [19,20]. The FoMV genome structure resembles that of PVX, the type species of the genus Potexvirus, and the gene functions are presumed to be similar as well [21,22]. The genome of FoMV contains five open reading frames (ORFs), and two subgenomic
promoters directing transcription of subgenomic RNAs (sgRNAs) 1 and 2 [21]. The genomic RNA allows the expression of ORF1 encoding the RNA-dependent RNA polymerase
(RdRP) with methyltransferase, helicase, and polymerase motifs in PVX [23]. The first sgRNA contains ORF2, 3 and 4 coding for the triple gene block (TGB) proteins
TGB1, TGB2 and TGB3, which are required for virus cell-to-cell movement [24]. The PVX ORF2 codes for a multifunctional protein that has RNA helicase activity,
promotes translation of viral RNAs, increases plasmodesmatal size exclusion limits,
and acts as a suppressor of RNA-mediated post-transcriptional gene silencing (PTGS)
[24]. The PVX ORF5 encodes the coat protein, which is required for viral encapsidation,
cell to cell movement, and long distance movement [25,26].

FoMV has a broad host range, infecting 56 species of the Poaceae and at least 35 dicot
species [27]. The sequence of FoMV genomic RNA was first published in 1991 [21]. Infectious full-length clones were constructed based on the same FoMV isolate and
some corrections to the published sequence were noted [28]. The significant difference between the gene organizations of FoMV and PVX is the
presence of ORF 5A upstream of the CP gene in FoMV. ORF 5A initiates 143 nts upstream
of the CP and extends the reading frame of CP gene. The 5A protein was produced in
vivo, but it was not required for either replication or productive infection of plants
[28]. Recently, the revised full-length sequence of foxtail mosaic virus clone was published
in 2008, and reveals a triple gene block structure similar to potato virus X[22].

The potexvirus replicase is the only protein translated directly from the full-length
genomic RNA, but other viral proteins are translated from 3' coterminal sgRNAs [29,30]. Two sgRNAs of approximately 2.1 and 0.9 kb in length have their 5' termini upstream
of the TGB and CP genes, respectively [19], while a 1.4 kb bicistronic (readthrough) sgRNA provides for the translation of TGB2
and TGB3 ORFs [31]. The integrity of the subgenomic promoter in a vector is very important for the accumulation
of sgRNA and target protein. However, the boundaries of sgRNA promoters have not been
delineated for FoMV.

Vectors using FoMV have not been reported until this present study, but foundational
work on potexvirus vectors has been completed with PVX. PVX was engineered to express
reporter genes cloned just upstream of the CP gene and expressed from a duplicated
copy of the CP subgenomic promoter [32]. The reporter gene was translated from a sgRNA separate from the other viral ORFs.
Because PVX has a linear helical capsid, rather than an icosahedral capsid, a recombinant
viral genome longer than wild type can still be encapsidated into infectious virus
particles. Expression of longer ORFs with this construct led to earlier deletion of
the ORF from the vector [33]. A PVX vector with a bicistronic sgRNA, carrying the reporter gene and the CP gene,
has also been constructed. An IRES site allowed for translation of the distal gene
on the sgRNA [34]. As mentioned previously, a deleted PVX vector has also been constructed [14]. A vector using another potexvirus, Alternanthera mosaic virus, has been recently developed [35]. In this vector system, the potexvirus was split into two components, the replicase
portion and the TGB/CP portion, each in its own binary vector and Agrobacterium culture. Following co-agroinoculation, recombination in vivo regenerates the full
length virus. In vivo expressed T7 RNA polymerase is used to transcribe the transgenic
virus components after agroinoculation.

In this study, a vector with the properties of high protein expression and greatly
lowered environmental risk was constructed. The TGB and CP genes of FoMV were removed
and replaced with heterologous sequences while the subgenomic promoter of the first
TGB gene (TGB1) was reserved to direct the transcription of the heterologous coding
sequence. The FoMV expression vectors driven by the 35S promoter were delivered as
a T-DNA to plant cells by agroinoculation. These severely crippled viral vectors would
have difficulty surviving in the wild, since they form no virions, use agroinoculation
for transmission, and are unable even to productively express marker genes unless
a silencing suppressor is supplied. However, expression levels are among the highest
of those reported from plant expression systems when silencing suppressor co-expression
is provided.

Results

Construction and agroinoculation of full length JL22/pFoMV

FoMV full length wild type viral cDNA from an in vitro transcription construct [26] was inserted into the binary vector, pJL22 [18] (Figure 1). To accomplish this, the PCR-amplified 5' end of the FoMV sequence was first transferred,
followed by the insertion of a restriction fragment containing the rest of the FoMV
sequence (Figure 1). The resulting construct (JL22/FoMV) comprised the 35S promoter, the full FoMV sequence,
a poly(A) tract present in the original sequence [28], and the 35S terminator.

Figure 1.Construction of pJL22/FoMV, the full length FoMV construct. (a) Source in vitro transcription construct of native FoMV [28]. (b) Recipient binary vector, pJL22 [18]. A PCR fragment with a phosphorylated blunt upstream end and a NotI site at the downstream
terminus was created by PCR from pFoMV10. This was ligated (1) into the StuI (blunt)
and NotI sites of pJL22 to create pJL22/FoMV5'. (c) Next, the remainder of the FoMV
sequence was added by creating a restriction fragment from pFoMV10 with PmlI/XbaI
digestion. This was ligated (2) to the same sites in pJL22/FoMV5' to create pJL22/FoMV
(Figure 3).

A. tumefaciens GV3101 was transformed with pJL22/FoMV and agroinoculated to N. benthamiana. At 1 and 2 weeks, there were no visible symptoms of viral infection on any plants.
To detect the presence of FoMV infection, total RNA was extracted from leaves and
screened by RT-PCR at 7 days post-inoculation (dpi). Systemic movement of FoMV was
demonstrated in inoculated plants by the presence of RT-PCR product, but no bands
were seen in uninoculated controls (data not shown). Thus, agroinfection with the
JL22/FoMV construct resulted in replication and movement of this FoMV sequence but
the infection proved very mild.

Construction and agroinoculation of deletion vectors (FECT)

Primers were designed to delete the TGB and CP genes while retaining the subgenomic
promoter of sgRNA1 and 3'-end of the CP gene to enable expression of foreign genes
(Table 1; Figure 2). The extent of the sgRNA1 promoter region was unknown, so the entire sgRNA1 putative
leader sequence and portions of the TGB1 ORF were included. Three upstream primers
were used to include the first 0, 22 and 40 bases of ORF of TGB1 to create pFECT0,
pFECT22 and pFECT40, respectively. The native TGB1 AUG was mutated to AUC and PacI
and AvrII sites were included as cloning sites. The 3' terminal part of CP FoMV gene
between AvrII and 3'- UTR was reserved because deletion of this region drastically
reduced vector accumulation in a deleted PVX vector [14]. The final form of these constructs is diagramed in Figure 3.

Figure 2.Deletions of pJL22/FoMV that led to the construction of the FECT vector series. Fragments containing various lengths of TGB1 subgenomic promoter were created by
PCR between an upstream primer binding in the FoMV replicase region (nt. 3044) and
a downstream primer which mutated the TGB1 AUG start codon to AUC and also added a
PacI and AvrII site downstream of the AUC. This fragment was digested with BamHI and
AvrII and inserted into pJL22/FoMV to take advantage of the native BamHI site in the
replicase (3081) and the AvrII site 93 bases upstream of the CP ORF translational
stop (stop at 6018) to create FECT0. FECT0 retains a subgenomic promoter consisting
of the replicase 3' end and the TGB1 RNA leader but has no TGB1 ORF codons; it also
retains 93 bases of the 3' end of the CP ORF. A PacI/AvrII cloning site is present
after the TGB1 leader in FECT0 and subsequent FECT versions. FECT22 and FECT40 extend
the potential TGB1 subgenomic promoter by an additional 22 and 40 bases, respectively,
of TGB1 upstream ORF sequence. These were created from FECT0 with the upstream primer
at 3044 and primers downstream of the AUC in FECT0. This fragment was digested with
native BamHI and added PacI (contained in the sequence of the downstream primers)
and inserted into these sites in FECT0.

Figure 3.Schematic diagram of (a) the T-DNA and (b) control regions of the FECT vector series. (a) The native FoMV infectious sequence [28] was inserted into a JL22 binary vector as in Figure 1 and is represented here as
"FoMV". The FECT series (Figure 2) constitutes the deleted FoMV vector. Various inserts
were placed into the PacI/AvrII cloning site of FECT40 in this study. p19 silencing
suppressor was added either in trans (via co-agroinoculation of a 35S/p19 construct)
or in cis (in the same binary vector as FECT40, but with a separate promoter and terminator).
Open boxes represent open reading frames; black stars: left border and right border
of T-DNA; block arrows: CaMV duplicated 35S promoter; black boxes: CaMV 3' terminator
sequence; gray arrows, subgenomic promoters; white arrow: nos promoter (Pnos); white
box: nos terminator (Tnos); dark grey boxes: Tobacco etch virus 5' non-translated
leader sequence (L); RB: T-DNA right border sequence; LB: T-DNA left border sequence;
TGB: triple gene block; CP, coat protein. (b) Three different lengths of TGB1 subgenomic
promoter were tested in their ability to drive GFP ORF expression. These were FECT0,
FECT22 and FECT40 which included sequence extending 0, 22 and 40 bases, respectively
from the first base of the TGB1 ORF. The start codon of TGB1 was mutated to ATC (underlined).
Restriction sites PacI and AvrII were introduced at the flank of GFP ORF as cloning
sites for other foreign inserts.

To test viral replication and foreign gene expression, GFP was inserted into each
of the FECT vectors (Figure 3). N. benthamiana was agroinoculated with GV3101 Agrobacterium cultures carrying FECT. At 2-4 days after agroinoculation, GFP-expressing cells could
be seen faintly using a hand-held UV lamp and fluorescence microscopy. At this time,
there were many faint green spots showing on leaves inoculated with FECT40/GFP and
FECT20/GFP (but fainter in the latter), but no green fluorescence could be detected
on leaves inoculated with FECT0/GFP (Figure 4a). Furthermore, the fluorescence was transient and, by eight days post-inoculation,
the GFP spots on all plants had disappeared (Figure 4b). Apparently, the transcription of agroinfiltrated T-DNA induced posttranscriptional
gene silencing (PTGS), which led to the inhibition of viral vector infection and the
reduction of viral productivity [36].

Figure 4.GFP expression with differing subgenomic promoters and rescue by gene silencing suppressor
coexpression. (a) 4 dpi and (b) 8 dpi. In both panels, the top row show fluorescence from N. benthamiana agroinoculated with FECT/GFP but without p19 silencing suppressor and the bottom row
shows leaves co-agroinoculation of FECT/GFP and 35S/p19. Only a small number of very
faint fluorescent spots were found in FECT22 and FECT40 leaves at 4 dpi (a), and this
fluorescence disappeared by 8 dpi (b). However, a vigorous fluorescence occurred with
the addition of 35S/p19. FECT0 plants did not fluoresce even in the presence of 35S/p19.
(c) FECT40/GFP was co-agroinoculated with 35S/p19 onto N. benthamiana and samples were taken each day of the time course and protein extracted by grinding
and centrifugation. Lanes: M, protein marker; 0, protein extract from uninoculated
leaf; 2 to 7, extracts from FECT agroinfiltrated leaves, 2 to 7 dpi, respectively.
(d) Western blot of GFP expression at 7 dpi. Lanes: 1, 0.2 μg GFP standard (E. coli generated); 2, FECT/GFP + 35S/p19 in N. benthamiana; 3, 35S/p19 alone in N. benthamiana.

Rescue with silencing suppressors

It has recently been demonstrated that co-inoculation of RNA silencing suppressor
proteins enhances the expression of heterologous proteins from viral vectors [14,18]. To test this effect, N. benthamiana plants were agroinfiltrated with a 1:1 mixture of 35S/p19 or 35S/HcPro and FECT/GFP
cultures. The accumulation of GFP was followed and imaged with a hand-held UV light
and fluorescence microscopy for 3-7 days post-inoculation.

When plants were co-infiltrated with the suppressor, the level of fluorescence was
surprising (Figure 4a and 4b). The fluorescence of the inoculated zones of FECT40/GFP plants was very clearly
seen under the UV lamp even with the room lights turned on (data not shown). FECT22/GFP
plants, though quite fluorescent, were clearly less so than FECT40/GFP inoculated
plants. Furthermore, no fluorescence was seen with FECT0/GFP with or without suppressor
co-infiltration (Figure 4a and 4b). Ds-Red was also expressed with FECT40 with similar results (data not shown).

Quantification and comparison to other vectors

The unusually high expression level led us to quantify the percent of total soluble
plant protein that the GFP represented in the inoculated zone. Over the course of
two weeks, fluorescence had appeared to increase through the first week and then stabilize
so a time course assay covering the first week was initiated. A. tumefaciens/FECT40/GFP + A. tumefaciens/35S/p19 co-infiltrated leaves from 2 to 7 dpi were homogenized and the relative amounts
of GFP in extracts of total soluble protein were measured with SDS-PAGE electrophoresis
and Coomassie blue protein staining (Figure 4c). GFP expression was detected from the second day after inoculation (Figure 4c). The expression level of fluorescent protein increased gradually, and stabilized
at 5-7 dpi (Figure 4c). GFP accumulated to 30% to 40% of the total soluble protein extracted, as measured
by densitometry.

This expression level was further quantified and compared to TMV vectors, which are
the most commonly used plant viral expression vectors. In a dilution comparison with
the full length TMV vector, JL24 [18], the expression of FECT40/GFP (top row) matched that of JL24 (bottom row) when both
were co-agroinoculated with p19 (Figure 5). Thus, even with the disadvantage of no systemic movement, FECT vector expression
was equivalent to that of an excellent systemic TMV vector. FECT40/GFP expression
was also equal to the TRBO-G vector [13], which is among the most strongly expressing of the deleted TMV vectors. In SDS-PAGE/Coomassie
blue densitometry analysis (Figure 6), FECT40 yielded GFP at 40% of total soluble protein (as for Figure 4c). The mg of GFP produced per gram fresh weight (gfw) of tissue was determined for
FECT40/GFP by comparison to a GFP standard and was 1.6-1.7 mg/gfw, similar to that
of TRBO-G with 1.3-1.7 mg/gfw (Figure 6). In comparison, the single-enhancer 35S promoter driving GFP expression in the standard
binary vector pGDG [37], induced by p19 expression, yielded GFP at only 0.03 mg/gfw, or more than 50-fold
less than FECT/GFP expression (Figure 6).

Figure 5.Dilution trial of agroinoculant with FECT and p19 on N. benthamiana plants. Top row: FECT40/GFP vector. Bottom row: JL24/GFP vector. A.t. cell suspensions were diluted, as noted in the figure, from an initial OD600 of
1.0. Pictures were taken under UV illumination at 4 dpi with photos taken at same
exposure.

In support of these values were calculations made by western blot analysis. The GFP
band of Figure 4d represented a 10 μl load from a 100× dilution of 300 μl of extract from 150 mg of
fresh leaf material. The protein content of the virus-expressed GFP band was estimated
as 0.2 μg by western blot, because it has the same density as the 0.2 μg GFP standard
generated by a bacterial expression system. From these data we again determined a
yield of 1.6 mg/gfw of GFP for FECT40/GFP. To further support this, GFP from FECT40/GFP
infected tissue from three replicates from another experiment was quantified by spectrophotometry
in comparison with bacterially-produced GFP standard. By this method, 1.58 ± 0.13
mg/gfw was determined for the GFP yield (data not shown).

Expression in monocots and legumes via agroinoculation

Since the natural host range of FoMV includes many grass and legume species, it was
appropriate to test GFP expression of FECT40/GFP plus 35S/p19 via agroinoculation
against a panel of grass and legume species via agroinoculation. Switchgrass, foxtail
millet, barley, wheat, oat and maize were co-agroinoculated with the mixture of two
Agrobacterium cultures containing FECT40/GFP and p19, respectively. Patches of widely spaced fluorescing
cells were observed by UV microscopy in all grass species (Figure 7a) beyond the few number of autofluorescent cells in uninoculated controls. However,
these isolated fluorescent cells were still quite uncommon in inoculated grass leaves.
A C58 strain of A. tumefaciens carrying FECT40/GFP was also tried, but did not provide any improvement in expression
rates in grass species. In the legume, M. trunculata, however, relatively larger numbers of isolated fluorescing cells were commonly encountered
in inoculated tissue (Figure 7b), while few or no autofluorescent cells were seen in uninoculated controls. Somewhat
fewer fluorescent cells were found in lentils, while no strong evidence for infected
cells was found in bean or cowpea by fluorescence microscopy.

Figure 7.Fluorescence microscopy of monocots and legumes agroinoculated with FECT40/GFP/p19. (a) Maize leaf infection. For all grasses tested, occasional areas of scattered
fluorescent cells were seen more commonly in FECT40/GFP/p19 agroinoculated leaves
than in p19 agroinoculated or noninoculated leaves, but such cells were rare nonetheless.
(b) Medicago trunculata leaf infection. Scattered fluorescent cells were common and FECT40/GFP/p19-infected
tissue was easily distinguishable from p19-inoculated control leaves under fluorescent
microscopy for both M. trunculata and lentils.

We have demonstrated in N. benthamiana that expression of FECT/GFP requires p19 co-expression and we expect that most cells
are doubly infected with agrobacteria containing FECT/GFP or 35S/p19. However, in
the agroinoculation of grasses, only a small fraction of cells, at best, are expected
to be even singly infected by Agrobacterium [38]. Even fewer cells would be expected to be doubly infected with two different agrobacteria
containing FECT/GFP and p19, respectively, and this might explain the difficulty in
visualizing fluorescent cells in grasses. For this reason, we added to the binary
vector carrying 35S/FECT/GFP another component carrying the nos promoter (Pnos) driving the expression of p19 and terminated by the nos terminator (Tnos). This cis-acting construct tested very strongly in N. benthamiana at 2 dpi (Figure 8), demonstrating an independence from p19 co-agroinoculation for strong fluorescence
in the cis construct. By 6 dpi, the fluorescence of the trans configuration (separate
binary vectors carrying FECT/GFP and 35S/p19) had increased so as to be somewhat superior
to the cis configuration. In spite of these N. benthamiana results, when the cis-configured FECT/GFP/p19 was co-agroinoculated onto various grass
species, no increase over the trans configuration in fluorescent cell numbers or brightness
was seen either by eye or by fluorescence microscopy.

Figure 8.Effect of p19 on GFP expression of FECT/GFP in trans and cis formats in N. benthamiana. GFP fluorescence at 2 dpi as seen under UV light for the standard 35S-driven FECT40/GFP
co-agroinoculated with 35S/GFP (trans) or for a binary vector containing both 35S/FECT40/GFP
and Pnos-driven p19 driven (cis). The cis format was constructed in an attempt to
better visualize FECT40/GFP expression in plants with poor rates of agroinoculation
(such as monocots), to eliminate the need for double infection by both p19 and FECT40/GFP.

Discussion

We have demonstrated very high protein expression rates in N. benthamiana for the deleted viral vector, FECT, in co-agroinoculation with 35S/p19. At 40% TSP
and 1.6 mg/gfw for the expression of GFP, FECT expresses well beyond expression rates
published for traditional plant virus vector systems [32,39]. The GFP expression of the best nonviral 35S system is 270-340 μg GFP/gfw aided by
p19 and 5-12 μg/gfw without p19 [36]. The most recently developed TMV vectors, TRBO [13] and the magnifection system [10] both report yields of up to 5 mg/gfw. In side-by-side comparison, with p19 co-inoculation
for both, however, FECT expressed GFP at levels equal to those of TRBO. Thus, we have
developed a simple and highly efficient protein synthesis vector system for plant-based
expression.

The FECT viral vector system has many features that enhance its environmental safety
when used via agroinoculation or potentially as a transgene. First, its genome is
reduced by almost one-third, making it severely crippled. Second, it does not replicate
efficiently unless the plant immune system is suppressed. Thus, if it were to somehow
infect another plant (e.g., via its Agrobacterium carrier), it would replicate poorly. Third, it lacks a coat protein and cannot form
a virion, making it improbable for the virus to survive outside of a host. Finally,
it is derived from a virus that in most hosts causes mild infections [27]. In fact, we observed no symptoms in N. benthamiana with the full length virus.

We observed a novel control mechanism operational with the FECT construct, which could
be very useful in further applications of FECT. In the absence of a silencing suppressor,
almost no expression of GFP occurred, and what expression did occur was transient,
disappearing by the seventh day after inoculation. This contrasts with the extreme
expression in the presence of the silencing suppressor. This was not the case with
the PVX vector of Komorova et al. [14]. Though PVX and FoMV are both potexviruses, the Komorova deletion vector performed
quite well, better than the full length standard PVX vector, even in the absence of
suppressor. Furthermore, this paper states that no extra bases of ORF were used to
extend the subgenomic promoter, but we found no expression when we emulated this with
FoMV. Only when extra bases of the TGB1 ORF were included was expression seen, and
for significant expression, suppressor co-expression was needed. Thus, the vectors
differed in design as well as performance.

Tyulkina et al. [40] examined the effect of removing the coat protein of TMV (tobamovirus family) and
portions of the movement protein. They found that complete removal of the movement
protein produced only small infection points, which could be rescued by the co-agroinoculation
of p19 or of various sized portions of the TMV movement protein. Thus, a similar effect
was found in the tobamoviruses as we found with the FoMV potexvirus. In contrast,
the work of Komorova et al. [14] appeared to clearly demonstrate that, for potexviruses, the removal of coat protein
and triple gene block genes produced a vector which was quite functional in the absence
of suppressor. Our results with the FoMV potexvirus were thus unexpected.

There may be functional differences in the replication mechanisms between FoMV and
PVX which are unexplored at present. For example, major differences in silencing suppressor
activity of TGB1 have been found between different potexviruses [41] and even between variant sequences of a single potexvirus, Alternanthera mosaic virus [42]. Though TGB1 was deleted in both the FoMV and PVX vectors, it may be that other features
in these viruses, affecting their ability to replicate when the TGB1 silencing suppression
is eliminated, may also exhibit taxonomic diversity. It may be further speculated
that host proteins that aid potexvirus replication, such as NbPC1P1 [43], might be more critical for some potexviruses than for others. The omission of, for
example, the CP gene, which NbPC1P1 binds to, might be more detrimental for FoMV than
for PVX.

The very low rate of infectivity of FECT in grasses is not unexpected. It is for this
reason that full virus vectors, capable of obviating low agroinfection rates via cell-to-cell
and long-distance movement, are used for agroinoculation studies [44,45]. There is only one paper to our knowledge that demonstrates successful visualization
of single cell agroinfections of grasses [38]. In this work, only scattered cells transformed with a 35S/GUS construct were visualized.
We were unable to utilize the GUS marker gene in this study because of GUS production
in the agrobacterium culture itself from the FECT/GUS construct (data not shown).
However, the creation of FECT/GFP/Pnosp19 opens the possibility of the use of FECT
as a viral transgene in grasses, since both p19 and FECT would be expressed in each
cell.

Conclusions

There are several potential applications for the use of FECT vectors. This system
has the capacity for high level expression of a variety of proteins, including GFP
(0.7 kb), the larger GUS (1.8 kb) and the multimeric Ds-Red (0.7 kb) proteins. The
inability of FECT to replicate significantly in the absence of silencing suppression
or to survive or infect in unencapsidated form greatly reduces environmental risk.
This system, then, would be expected to be amenable to the production of pharmaceutical
or industrial proteins via agroinoculation in the greenhouse. The tight on/off control
of this system also makes it suitable for an inducible transgenic system for field
use and for the expression of proteins toxic to the plant host. Finally, the use of
FECT as an agroinoculation vector for screening genes and gene variants for plant
expression before the construction of stably transgenic plants is facile due to high
expression and lack of cross contamination of greenhouses and growth chambers.

Vector construction

All FoMV viral cDNA constructs used in this study are derivatives of a wild-type FoMV
cDNA clone that was a gift from Nancy Robertson of the USDA [28] and were constructed with standard recombinant DNA techniques. The binary vector,
pJL22, provided by John Lindbo [18], has the mini binary plasmid, pCB301 [46], as a backbone. JL22 contains multiple cloning sites flanked by a 35S promoter and
35S polyA signal/transcription terminator (Figure 1). The source FoMV sequence had an additional 70 adenosine residues inserted after
the viral 3' terminus, followed by a XbaI site. To create JL22/FoMV, the 5' end of
FoMV was amplified by PCR with primers FoMV5'termUP and FoMV756NotDown and cut with
NotI. The 5' end of FoMV was cloned into JL22 digested with StuI and NotI to create
JL22/FoMV5'. The 3' end fragment of FoMV was prepared by restriction digest with PmlI
and XbaI and then inserted into JL22/FoMV5' also digested with PmlI and XbaI (Figure
1). pJL22 contains a CaMV 35S polyA signal to generate a poly(A) end, following the
poly(A) end already included in the Robertson sequence. This full viral cassette including
promoter and terminator is flanked with Left Border and Right Border of the T-DNA
("FoMV", Figure 3).

The FECT vector series was created from JL22/FoMV using PCR to delete the TGB and
CP genes. To ensure that the full sgRNA1 promoter was retained, primers were created
which included the first 0, 22 and 40 bases of ORF of TGB1 to create pFECT0, pFECT22
and pFECT40, respectively. PacI and AvrII sites were placed directly after the retained
sgRNA1 promoter of TGB1 for insertion of foreign ORFs. The downstream primer used
to make FECT0, namely, FoMV+0sgpDown, mutated the native TGB1 AUG start codon to AUC
and added both AvrII and PacI sites at 3' end of subgenomic promoter TGB1. Upstream
primer FoMVUp is upstream of a unique native BamHI site in FoMV (Figure 2). With these two primers, a PCR fragment was created and was digested with BamHI
and AvrII and cloned back into JL22/FoMV cut with BamHI and AvrII to create pFECT0.
Since a native AvrII site was present at nt. 5925, 93 bases upstream from the end
of the CP ORF (5371-6018) (Figure 2), inserting this PCR fragment into JL22/FoMV at the BamHI and AvrII sites enabled
all three TGB ORFs and most of the CP ORF to be deleted in one step to create FECT0.
The 3' terminal part of CP FoMV gene between AvrII and 3'- UTR was reserved for efficient
expression. To create vectors with longer subgenomic promoters, two primers, FoMV+22sgp
and FoMV+40sgp, were paired with FoMVUP to generate two PCR fragments including 22
and 40 bases of TGB1 ORF, respectively. These PCR fragments were digested and inserted
back into pFECT0 cut with BamHI and PacI to create pFECT22 and pFECT40. To insert
GFP, the GFP ORF was amplified using primers containing either PacI or AvrII sites
(Table 1) and the PCR product was digested and inserted into the PacI/AvrII cloning site in
FECT (Figure 3).

The high fidelity polymerase, Phusion (New England Biolabs (NEB), Beverly, MA), was
used according to company protocols in all constructions. Recombinant clones were
introduced into E. coli 10-beta electrocompetent cells (NEB, Beverly, MA) by electroporation at 1.44kV and
129 Ω for 5 ms using a BTX 600 Electro Cell Manipulator (BTX Inc., San Diego, CA,
USA) and colonies were screened by PCR using NEB Taq polymerase or by restriction
digests of plasmid minipreps prepared by Wizard Plus Miniprep Kit (Promega, Madison,
WI). Sequence verification was performed using a CEQ capillary sequencer (Beckman
Coulter, Fullerton, CA).

Agroinoculation

Agroinfiltration was performed as described [47] with modifications. Agrobacterium tumefaciens stain GV3101 was used for the agroinoculation of N. benthamiana and cereals. A. tumefaciens was transformed with plasmid constructs using the same conditions as for E. coli above. Agrobacterium transformants were selected at room temperature on Luria-Bertani plates containing
10 μg/ml rifampicin, 25 μg/ml gentamycin and 50 μg/ml kanamycin. A colony of A. tumefaciens was inoculated to 5 ml of L-MESA medium (LB media supplemented with 10 mM MES, 20
μM acetosyringone (Phytotechnology Labs, Shawnee Mission, KS)) and the same antibiotics,
and grown overnight at room temperature. The cells of the overnight culture were harvested
by centrifugation and resuspended in induction media (10 mM MES, 10 mM MgCl2, 100 μM acetosyringone) to a final OD600 of 1.0 and incubated for 2 h to overnight at room temperature. The cultures of A. tumefaciens were infiltrated into the underside of attached leaves with a 3 ml syringe without
needle. For co-agroinoculation of two or more bacterium cultures, cultures of A. tumefaciens were mixed in equal amounts and infiltrated together. The gene expression or virus
activity was tested at 6-8 days post-infiltration and one of three plant replicates
were analyzed per experiment.

RT-PCR

To detect FoMV (without GFP or DsRed) in the plant, total RNA was extracted after
7 dpi using Tri-Reagent (Sigma, St. Louis, MO) according to the manufacturer's protocol.
RT-PCR reactions were performed using the RT-PCR kit (NEB, Beverly, MA) as described
by the supplier. To detect the presence of virus sequence, FoMV specific primers were
used to amplify the partial viral genome.

Authors' contributions

ZL carried out the construction of the vectors, the inoculation of plants, protein
quantification, and drafted the manuscript. CMK conceived of the study, carried out
the vector comparison, cis-configuration and microscopy studies, performed the photography
and edited the manuscript. Both authors read and approved the final manuscript.